# Single‐Cell Transcriptomics Reveals Biomarkers for NK Cell Dysfunction in Endometriosis‐Associated Immune Dysregulation

## TL;DR

This study identifies key genes linked to NK cell dysfunction in endometriosis, offering potential biomarkers for diagnosis and treatment.

## Contribution

The study identifies GNLY, PRF1, and ENTPD1 as novel biomarkers for NK cell dysfunction in endometriosis.

## Key findings

- GNLY and PRF1 are predominantly expressed in NK cells and CD8+ T cells, correlating with activation signatures.
- ENTPD1 enhances migratory and invasive capacities of endometrial stromal cells and modulates NK cell function.
- A three-gene model shows good discrimination in training and validation cohorts for endometriosis diagnosis.

## Abstract

Endometriosis (EM) is associated with immune dysregulation, while dysfunction of natural killer (NK) cells is regarded as a key mechanism underlying immune escape and the persistent growth of ectopic lesions.

This study used single‐cell RNA sequencing (scRNA‐seq) on lesions from three patients with EM and on three normal endometrium samples and integrated these data with three bulk RNA‐seq datasets from GEO (GSE105765, GSE7305, and GSE6364). Seurat, Monocle, limma, least absolute shrinkage and selection operator (LASSO), and support vector machine recursive feature elimination (SVM‐RFE) were used for cell clustering, trajectory inference, differential expression analysis, and feature selection. Immune‐cell composition and pathway activity were evaluated with CIBERSORT and GSVA. Gene expression was validated by qPCR, and cell migration and invasiveness were assessed using wound healing and Transwell assays.

scRNA‐seq resolved 11 clusters assigned to eight major cell types. By integrating pseudotime features with bulk data, 20 differentially expressed genes (DEGs) were prioritized, and machine‐learning analyses identified three key genes: granulysin (GNLY), perforin 1 (PRF1), and ENTPD1. The three‐gene model showed good discrimination in the training set and two external validation cohorts (AUCs 0.84, 0.67, and 0.77, respectively). GNLY and PRF1 were predominantly expressed in NK cells and CD8+ T cells and correlated with activation signatures, whereas ENTPD1 was highly expressed in endometrial stromal cells and enhanced their migratory and invasive capacities. ENTPD1 may contribute to disease via adenosine signaling–mediated modulation of NK–cell function. In silico analyses also nominated candidate agents targeting this pathway, including resveratrol, ibuprofen, and danazol.

This study highlights the central role of NK–cell dysfunction in EM pathogenesis and proposes GNLY, PRF1, and ENTPD1 as potential molecular diagnostic biomarkers. Notably, ENTPD1 appears to have dual functions, including immunomodulation and promotion of stromal cell migration, which promotes lesion formation. These findings provide a mechanistic rationale and actionable targets for earlier screening and targeted therapy in EM.